V(D)J recombination constructs the variable regions of immunoglobulin and T cell receptor genes in developing lymphocytes through assembly of component gene fragments. The array of possible combinations for gene assembly is the primary basis for sequence diversity of the antigen binding receptor molecules in the immune system. Aberrant recombination reactions, such as those resulting in chromosomal translocations, can lead to lymphoid malignancies. In addition, reduced V(D)J recombination activity, as a result of point mutations in one or the other RAG protein, can lead to immunodeficiency diseases. To understand the molecular basis for these diseases, the factors that catalyze the V(D)J recombination reaction need to be better characterized. The initial site-specific DNA cleavage reaction is catalyzed by the V(D)J recombinase consisting of RAG1 and RAG2, proteins encoded by the recombination-activating genes. Together the RAG proteins bind to a conserved recombination signal sequence (RSS), which borders each gene fragment, and catalyzes double-stranded cleavage between the RSS and the bordering gene fragment in a two-step mechanism. The joining steps, resulting in assembly of the gene fragments, require additional ubiquitous factors including proteins that function in double-stranded DNA break repair. The broad objective of this proposal is to characterize the macromolecular assembly of the RAG proteins with the RSS. In our recent studies, we have identified structural domains of RAG1 that each either interacts with RAG2, the RSS, or the coding gene segments. Based on our results, we have developed a model for participation of the RAG1 DNA-binding domains at each step of the V(D)J recombination reaction. To test our model, we will further characterize the DNA-binding domains in RAG1, and determine their importance at each catalytic step in the recombination reaction. In addition, we will investigate potential regulatory roles for RAG2 in facilitating the association of RAG1 with the RSS. Finally, the requirement for participation of each RAG1 domain in the formation of the catalytically-active complex, as well as in DNA cleavage activity, will be tested. Results from these studies will provide a valuable framework in the determination of the assembly and mechanism of the V(D)J recombinase.